The present invention relates to a sensitive method for detecting drug-induced cytopenia mediated by antibodies that bind to blood cells or platelets only in the presence of a drug. Antibodies that react with blood cells or platelets only in the presence of an inciting drug are called "drug-dependent antibodies" (DDAb). This method also identifies the drug that is mediating the cytopenia in a particular patient.
A wide variety of medications have been implicated in the pathogenesis of drug-induced cytopenia (DIC). Cytopenia is a reduction or a lack of granulocytes, erythrocytes or platelets in the circulating blood. The particular cytopenias for which drug-dependent antibody binding can be identified using the method of the present invention include: granulocytopenia, also called agranulocytosis or neutropenia or leukopenia; thrombocytopenia; and hemolytic anemia.
Granulocytopenia is the clinical condition that exists when a patient has less than the normal number of granular leukocytes, usually neutrophils, in the blood. Thrombocytopenia is the clinical condition that exists when a patient has an abnormally small number of platelets in the circulating blood. Hemolytic anemia is a clinical condition in which the number of red blood cells in a patient's peripheral blood are less than normal due to abnormal destruction of erythrocytes in the body.
The main hindrance in diagnosing drug-induced antibody dependent cytopenia resides in the difficulty of objectively identifying the drug that is causing the cytopenia. The most widely used method of diagnosing drug-induced cytopenia is an indirect test that merely removes all medications that the cytopenic patient may be taking. If the patient's cytopenia resolves after drug removal, then the drug is said to be "associated" or "implicated" in causing the cytopenia.
Indirect implication of a drug in the pathogenesis of a patient's cytopenia is particularly problematic when a patient is on a regimen involving multiple medications, any one of which may be essential to treating a serious clinical condition. In vivo rechallenge with the suspected drug usually poses too great a potential risk. A method for identifying the one medication that is causing the cytopenia is needed so that the physician need not terminate administration of a cytopenic patient's needed, non-deleterious medications. Therefore, an in vitro test is needed to demonstrate the presence of an antibody in the patient's serum that binds to normal platelets, granulocytes or erythrocytes only in the presence of the offending drug, but not in the drug's absence.
More than 100 medications have been implicated in the pathogenesis of drug-dependent cytopenias, yet drug-dependent antibody binding has only been shown to be the pathogenetic mechanism underlying cytopenia in a few instances. These few instances include: (1) the detection of antibody binding to platelets which is dependent on the following drugs in the pathogenesis of thrombocytopenia: quinine, quinidine, acetaminophen, acetaminophen metabolites, Cefotetan, diphenylhydantoin, diazepam, novobiocin, penicillin, rifampin, vancomycin, valproic acid, cyanidanol, procainamide, sulfisoxazole, sulfamethoxazole, sulfamethoxazole metabolites, Para-aminosalicylic Acid (PAS), PAS metabolites and stibophen; Kelton et al., Blood 58:524 (1981); Cimo, et al., Am. J. Hematol. 2: 65 (1977); Hamilton et al., JAMA 239: 2586 (1978); Visentin et al., Transfusion 30:694 (1990); Kieffel, et al., Transfusion 27:262 (1987); and Aster et al., in Williams et al., (eds.) HEMATOLOGY (4th edition, McGraw Hill, 1990); (2) the detection of penicillin, Nafcillin, Oxacillin, propylthiouracil, propyphenazone and amodiaquine-dependent antibody binding to granulocytes in the pathogenesis of granulocytopenia; Salama et al., Br. J. Haematol. 72: 127 (1989); (Weitzman et al., Lancet 1: 1068 (1978); (Fibbe et al., Br. J. Haematol. 64: 363 (1986); (Salama et al., supra); (Rouveix et al., Br. J. Haematol. 71: 7 (1989); and (3) the detection of Ceftazidime, Cefotetan and diclofenac-dependent antibody binding to human erythrocytes in the pathogenesis of hemolytic anemia; Chambers et al., Am. J. Clin. Pathol. 95: 393 (1991); and Salama et al., Br. J. Haematol. 77: 546 (1991).
With the exception of the few instances of drug-dependent antibody binding to granulocytes, erythrocytes or platelets elucidated above, there has not been a reliable objective test for identifying drug-dependent antibody binding to granulocytes (G), erythrocytes (E) or platelets (P) (hereinafter referred to as GEP). The current assay systems have the major problems of resulting in false negative or false positive results. Some studies have reported results that suggest antibodies are not involved in a particular patient's cytopenia or in particular types of cytopenias. For example, no antibody binding could be demonstrated in a granulocytopenic patient who had been taking phenytoin. Sharafuddin et al., Acta Haematol. 86: 212 (1991). Also, in patients with clozapine-induced agranulocytosis, no drug-dependent antibody binding to granulocytes could be demonstrated. Pisciota et al., J. Lab. Clin. Med. 119: 254 (1992).
Prior art assays for the detection of antibody binding to granulocytes, such as the granulocyte immunofluorescence test (GIFT), are unable to demonstrate drug-dependent antibodies because of: (a) false negative results; (b) false positive results (no drug dependent binding of antibody); and (c) high background fluorescence. With many drugs, false negative results are probably due to the drug being unavailable in the reaction due to its insolubility in the buffer system. False positive results due to the drug alone increasing fluorescence are often the result of the insensitivity of prior art assays which required high concentrations of drug in order to detect drug-dependent antibody binding.
Thus, the diagnosis of drug-dependent antibody-mediated cytopenia has been difficult because there has not been a reliable in vitro method for detecting antibodies that are present in a cytopenic patient's serum and bind to blood cells or platelets only in the presence of a drug. A need exists for a reliable, objective method for detecting drug-dependent antibody binding to GEP in order to identify the particular drug causing a patient's cytopenia.